DESCRIPTION: The objective of this Phase II proposal is to complete the genetic development for a low cost, fermentation based, production method for thymidine. Thymidine, currently produced by chemical synthesis, is an important raw material for several anti-viral pharmaceuticals and other biomedical applications. Previous work at ChemGen to engineer E. coli for thymidine production established the technical feasibility. Several enzymatic steps of the de novo pathway for thymidine production have been engineered resulting in inducible non-inhibited thymidine production by dividing cells. In small scale fermentation, one strain produced 5.2 g/Liter thymidine from glucose. ChemGen proposes to complete the strain development by deregulating the first steps of the biosynthetic pathway utilizing Bacillus genes (cpa and pyrB) that code for enzymes not feed-back inhibited by pyrimidine nucleotides. Once regulated in concert with the rest of the system, this modification should effectively uncouple thymidine production from cell growth to allow commercial production. The technical innovation in this proposal lies in the first demonstration that an essential, long biosynthetic pathway, that is normally highly regulated, can be successfully modified for a commercial production process using a rational design. The final strain will contain at least seven genes foreign to the host. These genes code for enzymes with allosteric properties predicted to be favorable for thymidine production. The design includes genes from five sources, as well as a number of engineered host genes and chromosomal mutations. The investigators believe success will set a precedent for synthesis of worthwhile biochemical pharmaceutical intermediates by use of a rational approach to genetic modification and engineering of complex biosynthetic pathways. PROPOSED COMMERCIAL APPLICATION: AZT (Retrovir) is an important drug treatment for AIDS patients. The deoxyribonucleoside thymidine is the key chemical precursor, and single most expensive component of AZT manufacture. Thymidine currently sells for $500 to $600/Kg, but this new fermentation process is expected eventually to reduce the production cost of thymidine to less that $100/Kg. Other AIDS drugs, d4T and fluorothymidine, currently in trials are also produced from thymidine.